Major progress was made during the previous funding interval on both the RAG cleavage process and on the rejoining process, now called nonhomologous DNA end joining (NHEJ). The medical importance of the RAG cleavage process was illustrated by our discovery that RAG1 and 2 mutations can account for about 14 percent of human SCID (severe combined immune deficiency). In addition, many chromosomal translocations in lymphoid maligancies represent examples of some failure in the cleavage phase of V(D)J recombination. Lymphomas account for about 9 percent of neoplasia in adults and 50 percent in children under age 5. The rejoining phase is central to V(D)J recombination, but is of more general importance. The NHEJ process is used by all cells as the major pathway for repairing chromosome breaks resulting from pathologic causes, in addition to those resulting from V(D)J recombination. This proposal includes a systematic analysis of the biochemistry of each of the RAG cleavage steps and of each step in the NHEJ pathway (Aims 1 to 4). The binding, nicking, synapsis, and hairpin formation by the RAG complex is examined. Among the key issues is the basis for the 12/23 rule. The mechanism and regulation of hairpin opening is examined, as is the transition from the cleavage phase to the rejoining phase. This transition involves Ku and the 469 kDa DNA-dependent protein kinase (DNA- PK). After the transition to the NHEJ pathway, the coding ends must be processed to make them compatible. Our studies include identification and analysis of these processing enzymes. Recent work indicates that additional proteins in the NHEJ pathway have yet to be identified. Understanding this pathway is central to V(D)J recombination, but also has much broader significance for issues of genetic stability and cancer. Aim 5 utilizes all of these assays to understand human SCID defects due to V(D)J recombination. Aim 6 is directed at continuing our earlier work on chromatin effects on V(D)J recombination.